The DC Generation Medium DXF (C-28052) is recommended for freshly isolated monocytes or mononuclear cells. When using this medium, cells will attach efficiently, so that an additional medium like the Monocyte Attachment Medium (C-28051) is not necessary.
In contrast, when using the DC Generation Medium (C-28050) for freshly isolated monocytes or mononuclear cells, the Monocyte Attachment Medium (C-28051) is needed for an efficient attachment.

Mononuclear cells are mostly used in immunology, infection biology, hematology and cancer research to study subpopulations of blood cells.
Our Mononuclear Cell Medium (C-28030) is intended for short-term maintenance (up to 48 hrs) of the thawed hMNC before you proceed with your experiments. The number of PDs will depend on the subsequent cell culture conditions and is not determined by PromoCell.
Please note: Depending on the conditions, the ratio of the subpopulations will gradually change, as the different blood cell types behave in different manners. Researchers normally start soon after thawing to either select the cell type of their interest (e.g. hematopoietic cells, endothelial progenitor cells) or perform experiments with all populations of hMNCs (e.g. to study effects on toxicity, viability or metabolism).

It is not recommended to leave the blood cells in the Monocyte Attachment Medium for longer than 1.5-2 hrs. The medium was developed for (short-term) attachment of the monocytes and does not provide nutrients for a longer time period. Leaving the cells in Monocyte Attachment Medium for a longer time or even overnight will induce apoptosis and lead to the loss of the cells.
If necessary, you can reduce the incubation time to 1 hr. In this case, it is advisable to equilibrate the media in the incubator before so that you can immediately and directly add the appropriate amount of PBMC suspension.

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Short protocol:
– Refer to the cell number per vial given in the lot-specific CoA and calculate the needed media volume to result in 1 x 106 hMNC/ml. Transfer the needed volume in a cell culture vessel and equilibrate in the incubator (37°C, 5% CO2) for 30 minutes
– Remove the cryovial from liquid nitrogen and immediately place it on dry ice, even for short transportation
– Submerge the vial into a 37°C water bath for 2 min, then rinse it with 70% ethanol and open it under the laminar flow bench. Use a 2 ml serological pipette to transfer the cells into the tissue culture vessel containing the prewarmed medium (e.g. Mononuclear Cell Medium, C-28030) without resuspension
– Place the vessel back in the incubator
Note: Do not resuspend the cells at any time, since clumping may occur. For complete recovery, leave the cells untouched for at least 18 hours. Change the medium after 18-24 hours.

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The PromoCell DC Generation Media have been developed for the easy and efficient generation of immature as well as fully mature myeloid dendritic cells from peripheral blood monocytes.For freshly isolated mononuclear cells (MNC) and monocytes we recommend our DC Generation Medium DXF (C-28052), for cryopreserved monocytes our DC Generation Medium (C-28050). When using DC Generation Medium (C-28050) with fresh MNC, the Monocyte Attachment Medium (C-28051) is needed in a first step for efficient adherence of the monocyte fraction.
Please see Instruction Manual, Application Note and the graph below for further details.

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The Monocyte Attachment Medium allows for the efficient adherence selection (within 1 hr) of monocytes from freshly isolated mononuclear cells while maintaining optimal cell health. The Time-consuming and costly immunomagnetic purification of monocytes prior to DC generation is not necessary and can be skipped when using this medium.
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